Hemoglobin is a metalloprotein of red blood cells and is one of the major constituent of whole blood which is responsible for transporting oxygen in mammals. Hemoglobin primarily exists in five different forms namely, oxy-hemoglobin, deoxy-hemoglobin, methemoglobin, carboxy-hemoglobin and sulfhemoglobin. Only oxy-hemoglobin and deoxy-hemoglobin are involved in oxygen transport; they interconvert into each other in the process of oxygen transportation. Methemoglobin, the oxidized form of hemoglobin in which the iron molecule remains in +3 oxidation state is incapable of transporting oxygen. Carboxy-hemoglobin and sulf-hemoglobin, forms in which iron molecule stays in +2 oxidation state, are also incapable of transporting oxygen since their oxygen binding site stays occupied by strong ligands, CO and S respectively.
Estimation of Hemoglobin is among the most frequent blood tests in pathological practices globally. Total Hemoglobin in whole blood is defined as an algebraic sum of all five forms. Measuring total hemoglobin accurately and in a cost effective manner is a challenging task primarily due to three reasons. First, different forms of hemoglobin have different oxidation states and hence they all cannot be quantified using any single redox reaction. Second, different forms of hemoglobin have different and overlapping optical absorbance spectra with different molar extinction coefficient. Third, due to their high stability, carboxyhemoglobin and sulfhemoglobin do not react readily with most of the reactants, including oxidants and substituents, and hence their optical spectral interference with any hemoglobin derivatives is practically unavoidable. The predominant optical interference in hemoglobin quantification is offered by carboxy-hemoglobin as sulfhemoglobin at maximum is found in traces in whole blood.
The state-of-the-art methods for estimation of total hemoglobin levels can technically be categorized under three categories, discussed as follows: Firstly, the use of pathological blood analyzers, spectrophotometry of unaltered lysed blood at different wavelengths. These analyzers quantify each form of hemoglobin independently by measuring optical absorbance of the blood sample at the peak absorbance wavelengths of each form of hemoglobin. The recorded readings are then interpreted by specialized computing algorithms. Such analyzer systems are highly expensive as they require complex and expensive optical & computing systems.
PCT International Publication No. WO 03/056327 A1 discloses a method for quantitative hemoglobin determination in undiluted unhemolysed whole blood which involves performing a first absorption measurement at a wavelength in the range of 490-520 nm directly on the sample in the cuvette, and further conducting a second absorption measurement, and processing results of first and second absorption measurements to determine the concentration of hemoglobin in the sample.
Spectroscopic method and apparatus for total hemoglobin measurement is disclosed in U.S. Pat. No. 7,449,339B2.
Second method uses non-invasive optical meters, multi-wavelength transmittance method measure transmittance of light of different wavelengths through a part of live body (usually finger tips). Conceptually and operationally similar to electronic non-invasive SpO2 meters, these non-invasive devices measure transmittance at peak absorbance wavelengths of different forms of hemoglobin and measure the percentage of different forms accurately. These percentages values are multiplied with an average hematocrit value to arrive at the absolute concentrations of different forms of hemoglobin. This method is highly error prone as it heavily depends on the assumed hematocrit value, which is different in different samples. As disclosed in U.S. Pat. No. 5,277,181 A entitled “Noninvasive measurement of hematocrit and hemoglobin content by differential optical analysis relates to the noninvasive measurement of blood hematocrit and hemoglobin content using differential optical absorption of two or more wavelengths of light.” Also, U.S. Pat. No. 5,692,503 discloses a method for noninvasive (in-vivo) total hemoglobin, oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin and methemoglobin concentration determination.
Third method is based on hemoglobin chemistry. All forms of hemoglobin are converted either into hemochromogens or hemoglobin derivatives by the action of surfactants or chemicals respectively. The finally developed complex is then quantified optically by measuring its peak absorbance value and correlating that value with concentrations of hemoglobin. It has been experimentally observed that due to very high stability carboxy-hemoglobin remains unaffected by the actions of used reagents. Moreover, carboxy-hemoglobin's optical absorbance spectrum interferes with the spectra of most of the hemoglobin derivatives. With varying concentration of carboxy-hemoglobin, this interference of carboxyhemoglobin leads to inaccuracy in results while quantifying the complex at its peak absorbance wavelength.
Use of reagents in hemoglobin determination have been disclosed in U.S. Pat. Nos. 5,834,315, 3,874,852, 4,800,167, 4,853,338 and US Publication no. US2003/0044995 A1.
As described in U.S. Pat. No. 5,692,503 (A), wavelengths of one or more analytes is selected in the range of 480 nm to 630 nm, with separate analyte wavelength for each hemoglobin species. Thereafter the measurement of individual absorbance of each species is solved for determining the total concentration of hemoglobin.
Devices based on the third method currently follow primarily three approaches described as follows. First approach prefers converting all forms of hemoglobin into hemochromogens by addition of high concentration (above the critical micelle concentration) of detergents and measuring absorbance at the peak absorbance wavelength of hemochromogen. Such approach fails to address the carboxyhemoglobin interference problem. Moreover, since this approach requires measurement of absorbance of blood in reagents coated cuvettes, it requires a large amount of blood sample for testing.
Second approach prefers converting different forms of hemoglobin into a common specie by converting them into a hemoglobin derivative, for example, azide-methemoglobin, and measuring the absorbance or reflectance at the peak absorbance wavelength of the derivative. These methods too fail to convert to the carboxyhemoglobin form to common specie due to highly stability of this form and its resistance to reactivity with most of the chemical reactions.
Use of above-mentioned reagents has been disclosed in STAT-Site MHgb Test System (Stanbio Laboratory Co., Boerne, Tex.), which measures hemoglobin by using a reflectance meter and a hemoglobin test card wherein the test reaction is based on aazidemethemoglobin method. The HemoCue system (HemoCue Inc., Cypress, Calif.) carries a test reaction consisting of sodium deoxycholate, sodium nitrite, and sodium azide nitrite which lyses the blood and converts hemoglobin to hemiglobinazide.
The third approach prefers measuring absorbance or reflectance of lysed blood in its natural state at 523 nm, the natural isobestic point of all four forms of hemoglobin. This method stays under a risk of low sensitivity and poor signal to noise ratio due to three reasons. First, the molar extinction coefficient of different forms of hemoglobin at 523 nm is low (nearly 50% of extinction coefficient of oxy-hemoglobin at its peak absorbance wavelength, 540 nm), resulting in low sensitivity of the device and hence inability to differentiate between different concentrations of hemoglobin. Second, the method is susceptible to low signal to noise ratio as the spectrum of methemoglobin at 523 nm has no clearly differentiated peak and is almost parallel to the abscissa. Third, it has experimentally been found that the absorbance spectra of carboxyhemoglobin and oxyhemoglobin stay close to each other at 523 nm but do not coincide or interest each other at this wavelength. The Hemoglobin test strip and Analysis system disclosed in U.S. Pat. No. 7,379,167 B2 uses an apparatus, which emits light at 522 nm for measurement of hemoglobin concentration. This phenomenon also adds to the inaccuracy of the device.
In light of the above drawbacks in the state-of-the-art there is a need to provide a device and method for blood hemoglobin measurement without carboxyhemoglobin interference thereby providing an accurate result, which addresses the limitations of the state-of-the-art.